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. 2023 Dec 13;8(51):49228–49243. doi: 10.1021/acsomega.3c07216

Biooriented Synthesis of Ibuprofen-Clubbed Novel Bis-Schiff Base Derivatives as Potential Hits for Malignant Glioma: In Vitro Anticancer Activity and In Silico Approach

Muhammad Ayaz †, Aftab Alam †, Zainab ‡, Mohammad Assad §, Aneela Javed ∥, Mohammad Shahidul Islam ⊥, Huma Rafiq ∥, Mumtaz Ali †,*, Waqar Ahmad †, Ajmal Khan #, Abdul Latif †, Ahmed Al-Harrasi #,*, Manzoor Ahmad †,*
PMCID: PMC10764114  PMID: 38173864

Abstract

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This research work is based on the synthesis of bis-Schiff base derivatives of the commercially available ibuprofen drug in outstanding yields through multistep reactions. Structures of the synthesized compounds were confirmed by the help of modern spectroscopic techniques including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1H NMR, and 13C NMR. The synthesized compounds were evaluated for their anticancer activity using a normal human embryonic kidney HEK293 cell and U87-malignant glioma (ATCC-HTB-14) as a cancer cell line. All of the synthesized compounds among the series exhibited excellent to less antiproliferative activity having IC50 values ranging from 5.75 ± 0.43 to 150.45 ± 0.20 μM. Among them, compound 5e (IC50 = 5.75 ± 0.43 μM) was found as the most potent antiprolifarative agent, while 5f, 5b, 5a, 5n, 5r, 5s, 5g, 5q, 5i, and 5j exhibited good activity with IC50 values from 24.17 ± 0.46 to 43.71 ± 0.07 μM. These findings suggest that these cells (HEK293) are less cytotoxic to the activities of compounds and increase the cancer cell death in brain, while the lower cytotoxicity of the potent compounds in noncancerous cells suggests that these derivatives will provide promising treatment for patients suffering from brain cancer. The results of the docking study exposed a promising affinity of the active compounds toward casein kinase-2 enzyme, which shows green signal for cancer treatment.

Introduction

Uncontrolled cell growth causes cancer, which is the deadliest noninfectious disease. Cancer is a serious threat to humanity and ranks as the second leading cause of death worldwide.1 Although chemotherapy is a commonly used cancer treatment, drug resistance and severe adverse effects remain significant challenges associated with anticancer therapy.2 To overcome these challenges, researchers are developing new compounds with multitarget inhibitory properties.3 Of particular interest is the overexpression of enzyme cyclooxygenase-2 (COX-2) in pancreatic, breast, colorectal, stomach, and lung carcinomas, making COX-2 a prime target for the synthesis of new anticancer agents.4,5

According to 2020 reports, there were 19.3 million cases of cancer and 10 million cancer-related deaths.6,7 The United States of America (USA) witnessed an estimated 1.9 millions new cancer cases and 0.6 million cancer related deaths in the year 2022.8 In the future, cancer incidence is expected to increase, with an estimated 26 million cases predicted in 2030.9 There exists a significant disparity between the supply and demand of new anticancer drugs.10 Current anticancer agents have notable post-treatment adverse effects because they lack selectivity.11,12 Consequently, there is an urgent need to invent and develop new selective anticancer therapies that cause fewer side effects and have multiple modes of action.9,13 The link between inflammation and cancer was first suggested by Rudolf Virchow who noticed the presence of leukocytes in tumor cells.14,15 It is now widely recognized that chronic inflammation increases the risk of cancer. During the inflammation process, various growth factors, including epidermal growth factor (EGF) and fibroblast growth factor (FGF), are released, which stimulate cell proliferation and in turn cause cancer.16,17 The biosynthesis of prostaglandins during inflammation is primarily facilitated by the enzyme cyclooxygenase (COX). COX exists in three isoforms, COX-1, COX-2, and COX-3. COX-1 is present in normal tissues and regulates prostaglandin production, which is necessary for normal physiological function. On the other hand, COX-2 is produced during inflammation and carcinogenesis.18,19 Nonsteroidal anti-inflammatory drugs (NSAIDs) block COX-1 and COX-2 enzymes to varying degrees, reducing prostaglandin biosynthesis.20,21 However, COX-1 blockage leads to several complications, the most common of which is gastrointestinal ulceration.22,23 COX-2 is the primary inflammatory mediator, and its expression is upregulated during inflammation.24 As a result, COX-2 has been the target of anti-inflammatory drugs for many years.25,26 The COX-2 enzyme plays a critical role in cancer initiation, since it inhibits apoptosis and initiates angiogenesis. An increase in COX-2 levels has been observed in various cancers, including colorectal (60%), breast (40%), pancreatic, esophageal, lung cancer, and melanoma.27 NSAIDs such as ibuprofen (1), naproxen (2), diclofenac (3), piroxicam (4), and celecoxib (5) are frequently used to manage cancer-related pain and inflammation (Figure 1). Inhibiting COX-2 through NSAIDs has been found to impede the cancer process and promote metastasis.28 Therefore, COX-2 has emerged as a potential target for new anticancer drugs, and there is increased research in this direction.29 Celecoxib (5), a selective COX-2 inhibitor, has demonstrated anticancer properties against certain cancers, such as ovarian cancer and adenomas.30 There is currently significant research into discovering and synthesizing new COX-2 inhibitors due to their unique role in cancer chemotherapy.

Figure 1.

Figure 1

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Selective and nonselective NSAIDs (1–5).

The most commonly utilized therapeutic drugs worldwide are NSAIDs (nonsteroidal anti-inflammatory drugs).31 Ibuprofen is the most commonly used medication that inhibits cyclooxygenase enzymes like COX-1 and COX-2, which results in the reduction of prostaglandin production, leading to a decrease in inflammation and pain mediators.32 In addition to decreasing or releasing pain, ibuprofen and other NSAIDs have been shown to be useful as anticancer agents in various kinds of cancers. Previous clinical and epidemiological research has indicated that regular and prolonged usage of ibuprofen may be effective in treating and reducing the risk of 7–10 different types of cancer, including the four major types: colon, breast, lungs, and prostate cancer.33−35 But long-term use of NSAIDs can cause various side effects such as ulcers,36 gastrointestinal bleeding, and heart attacks.37 These adverse effects are caused by the free carboxylic group that is present.38

Hydrazone-Schiff base derivatives have gained recognition for their wide range of pharmacological activities.39,40 Among them, various hydrazones have been employed as effective anticancer agents. İhsan Han et al. synthesized naproxen-based hydrazone-Schiff bases that showed the most potent anticancer activity against human breast cancer cell lines (MDA-MB-231 and MCF-7).41 Various hydrazone and bis-hydrazone compounds containing a sulfonate moiety were synthesized and screened against various cancer cell lines; compounds 7 and 8 are more potent against MCF-7 cell line42 (Figure 2).

Figure 2.

Figure 2

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Different hydrazone-Schiff base derivatives (6–9) having anticancer activities.

Casein kinase-2 (CK2) is a ubiquitous serine/threonine protein kinase that plays a key role in the regulation of a wide range of cellular processes. Recent studies have suggested that CK2 may be involved in cancer development and progression.43 CK2 is the most pleiotropic of all protein kinases with more than 300 substrates implicated in a wide variety of cellular functions in gene expression, signal transduction, proliferation, and cell survival. Antisense oligonucleotides against CK2 or CK2ß, microinjection of anti-CK2 antibodies, or inhibitors of CK2 inhibit cell cycle progression. Elevated CK2 activity has been demonstrated in a number of cancers and has been shown to regulate the activity of various oncoproteins and tumor suppressor proteins. Development of CK2 inhibitors is ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds.44 Casein kinase-2 (CK2) is an oncogenic protein kinase that contributes to tumor development, proliferation, and suppression of apoptosis in multiple cancer types.45

Experimental Section

Materials and Methods

All of the chemicals, reagents, and solvents consumed in this research work were analytical-grade (99% pure) and purchased from TCI, Merck, Sigma-Aldrich, and the starting material ibuprofen was obtained from Sigma-Aldrich (CAS No: 51146-56-6). Thin layer chromatography (TLC) was performed using precoated aluminum silica gel plates (Kieselgel 60, 25, E. Merck, Germany) and a solvent system consisting of ethyl acetate and n-hexane. The visualization was done under UV light at 254 and 365 nm. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) spectra were recorded on a mass spectrometer (Waters Quattro Premier XE, Waters, Milford, MA). Nuclear magnetic resonance (NMR) spectra were conducted on a Bruker NMR spectrometer (Zurich, Switzerland) operating at 600 MHz for 1H NMR and 150 MHz for 13C NMR. The internal standard used in NMR spectra is tetramethylsilane (TMS), and chemical shift values are reported in parts per million (ppm). Coupling constants (J) are given in Hz, while multiplicities are expressed as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), quartet (q), and multiplet (m). Melting points (mp) for the synthesized ibuprofen derivatives were determined using the Stuart SMP10 melting point apparatus.

Procedure for the Synthesis of 2-(4-Isobutyl phenyl)propanehydrazide (2)

The ibuprofen acid (12.1 mmol, 2500 mg) was first activated with the help of 1,1-carbonyldiimidazole (CDI) (14.5 mmol, 2350 mg) in the presence of catalytic amount of triethyl amine (TEA) in 25 mL of tetrahydrofuran (THF) solvent, and then 2 mL of hydrazine hydrate was added and refluxed for 3–4 h. The reaction progress was monitored by TLC (n-hexane/ethyl acetate 6:4). After the reaction mixture was complete, it was poured onto crushed ice. The resulting precipitates were filtered, washed with distilled water, and dried in air. The products were recrystallized in ethanol.

White amorphous solid: Yield: 93%, 2.47 g; mp: 76–77 °C; 1H NMR (600 MHz, CDCl3): δ 9.05 (t, J = 4.7 Hz, 1H, −NH), 7.26–7.19 (m, 2H, Ar–H), 7.06 (d, J = 7.5 Hz, 2H, Ar–H), 4.11 (d, J = 4.6 Hz, 2H, –NH2), 3.75 (q, 1H, −CH−), 2.53–2.40 (m, 2H, −CH2−), 1.84 (m, 1H, −CH), 1.40 (d, J = 6.9 Hz, 3H, −CH3), 0.85 (d, J = 6.8 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 173.41 (–C=O), 142.23 (Ar–C), 141.75 (Ar–C), 129.94 (2Ar–C), 127.37 (2Ar–C), 45.80 (−CH2−), 45.64 (CH−), 30.81 (−CH−), 22.84 (2 −CH3), 18.60 (CH3). HRMS (ESI+): 220.1576 [M + H]+ calcd for C13H20N2O: 220.1586.

Procedure for the Synthesis of Ethyl-2-(4-((2-(2-(4-isobutyl phenyl)propanoyl)hydrazono)methyl)phenoxy)acetate (3)

Compound 2 (11.1 mmol, 2450 mg) was dissolved in ethanol (20 mL) and an excess of hydrazine hydrate was added, and the mixture was refluxed for 4–5 h; after completion, the reaction mixture was poured in ice and formed precipitate. The precipitates were filtered and washed with cold water to give the title compound 3.

White amorphous solid: Yield: 90%, 1660 mg; mp: 161–163 °C; 1H NMR (600 MHz, CDCl3): δ 9.15 (br.s, 1H, –NH), 7.65 (s, 1H, –NH), 7.56 (s, 1H, –N=CH−), 7.30 (d, J = 8.0 Hz, 2H, Ar–H), 7.24 (d, J = 8.0 Hz, 2H, Ar–H), 7.12 (d, J = 8.0 Hz, 2H, Ar–H), 7.05 (d, J = 7.8 Hz, 2H, Ar–H), 4.63 (br.s, 2H, –NH2), 4.60 (s, 2H, –OCH2), 2.42 (t, J = 7.2 Hz, 2H, –CH2), 1.80 (m,1H, −CH), 1.28 (d, J = 6.8, 3H, −CH3), 0.88–0.86 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.4 (C=O), 168.5 (C=O), 159.5 (Ar–C–O), 147.2 (–N=CH), 142.2 (Ar–C), 140.16 (Ar–C), 137.4 (Ar–CH), 129.8 (Ar–CH), 129.6 (Ar–CH), 129.1 (Ar–CH), 128.6 (Ar–CH), 127.6 (Ar–CH), 127.3 (Ar–CH), 114,4 (2Ar–CH), 65.3 (−OCH2), 45.0 (−CH), 44.9 (−CH2), 30.1 (−CH), 22.3 (2-CH3), 18.2 (−CH3). HRMS (ESI+): 396.2140; [M + H]+ calcd for C24H28N4O3: 396.2145.

Procedure for the Synthesis of N′-(4-(2-Hydrazinyl-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (4)

A mixture of 2-(4-isobutyl phenyl)propanehydrazide (3) (4.1 mmol, 1650 mg) and ethyl-2-(4-formylphenoxy)acetate (1620 mg, 4.1 mmol) in ethanol (20 mL) in a catalytic amount of glacial acetic acid was refluxed for 4 h. After completion, the reaction mixture was poured onto crushed ice. The resulting precipitates were filtered, washed with distilled water, dried in air, and recrystallized from ethanol to give the title compound 4.

Yellowish amorphous solid: Yield: 88%, 1.25 g; mp: 114–116 °C; 1H NMR (600 MHz, CDCl3): δ 8.63 (br.s, 1H, –NH), 8.23 (s, 1H, –N=CH−), 7.60–7.54 (m, 2H, Ar–H), 7.30 (d, J = 8.0 Hz, 2H, Ar–H), 7.16 (m, 1H, Ar–H), 7.16–7.11 (m, 1H, Ar–H), 7.07 (d, J = 8.0 Hz, 2H, Ar–H), 6.90 (d, J = 9.0 Hz, 1H, Ar–H), 4.66 (s, 2H, –OCH2), 4.28–4.23 (m, 2H, –OCH2), 3.66–3.55 (m, 1H, −CH), 2.45 (d, J = 7.0 Hz, 2H, –CH2), 1.86–1.76 (m, 1H, 1H, −CH), 1.50 (d, J = 6.8 Hz, 1H, −CH), 1.28 (t, J = 6.6 Hz, 3H, −CH3), 0.89–0.83 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.0 (C=O), 168.5 (C=O), 159.2 (Ar–C–O–CH2), 147.2 (Ar–C), 142.2 (–N=CH−), 141.01 (Ar–C), 140.16 (Ar–C), 138.4 (Ar–CH), 129.8 (Ar–C), 129.6 (Ar–CH), 129.2 (Ar–CH), 128.6 (Ar–CH), 127.5 (Ar–C), 65.3 (−OCH2), 61.3 (−OCH2−), 45.0 (−CH), 44.9 (−CH2), 30.1 (−CH), 22.3 (2-CH3), 18.2 (−CH3), 14.1 (−CH3). HRMS (ESI+): 396.2161 [M + H]+ calcd for C22H28N4O3: found 396.2176.

Procedure for the Synthesis of Compounds 5a–t

Ibuprofen bis-hydrazide 4 (0.25 mmol, 90 mg) and substituted benzaldehydes (0.25 mmol) (Table 1) were dissolved in ethanol (10 mL) in a round-bottomed (RB) flask (250 mL). Glacial acetic acid (3–5 drops) was added into the reaction mixture and refluxed for 3–4 h. The reaction progress was monitored by TLC (n-hexane/ethyl acetate = 7:3). After completion, the reaction mixture was poured onto crushed ice. The resulting precipitates were filtered, washed with distilled water, and dried in air. The products were recrystallized in ethanol.

Table 1. Different Aromatic Substituents of Ibuprofen bis-Hydrazone (5a–t).

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Spectral Interpretation of the Synthesized Products 5a–t

2-(4-Isobutyl phenyl)-N′-4-(2-(2–4-nitrobenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)propanehydrazide (5a)

Off white amorphous solid: Yield: 90%, 118 mg; mp: 185–187 °C; 1H NMR (600 MHz, CDCl3): δ 9.41 (br.s, 2H, 2-NH), 8.47 (s, 2H, 2-N=CH−), 8.24 (d, J = 9.0 Hz, 2H, Ar–H), 7.80–7.73 (m, 4H, Ar–H), 7.27 (d, J = 8.4 Hz, 2H, Ar–H), 7.14–7.12 (m, 2H, Ar–H), 7.06 (d, J = 7.8 Hz, 2H, Ar–H), 4.68 (s, 2H, −CH2), 4.64–4.62 (m, 1H, −CH), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.80–1.75 (m, 1H, −CH), 1.53 (d, J = 7.2 Hz, 3H, −CH3), 0.84–0.82 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.9 (C=O), 170.2 (C=O), 148.4 (Ar–C–NO2), 141.3 (Ar–C), 140.4 (2-N=CH), 140.0 (Ar–C), 139.8 (Ar–C), 138.1 (Ar–C), 130.0 (2Ar–CH), 129.6 (2Ar–CH), 128.0 (2Ar–CH), 127.6 (2Ar–CH), 127.0 (Ar–C), 124.1 (2Ar–CH), 123.9 (2Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.6 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.4 (−CH3). HRMS (ESI+): 545.2675 [M + Na]+ calcd for C29H31N5NaO5: 545.2724.

2-(4-Isobutyl phenyl)-N′-4-(2-(3-nitrobenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)propanehydrazide (5b)

Brownish amorphous solid: Yield: 88%, 116 mg; mp: 163–165 °C; 1H NMR (600 MHz, CDCl3): δ 9.41 (br.s, 2H, 2-NH), 8.46 (s, 2H, 2-N=CH−), 8.21–8.17 (m, 2H, Ar–H), 7.87 (d, J = 7.2 Hz, 2H, Ar–H), 7.73 (br.s, 1H, Ar–H), 7.54 (t, J = 7.8 Hz, 1H, Ar–H), 7.30 (d, J = 7.8 Hz, 2H, Ar–H), 7.16–7.13 (m, 1H, Ar–H), 7.10 (d, J = 7.8 Hz, 3H, Ar–H), 4.68 (s, 2H, −CH2), 4.63–4.59 (m, 1H, −CH), 2.38 (d, J = 7.2 Hz, 2H −CH2), 1.83–1.75 (m, 1H, −CH), 1.53 (d, J = 7.2 Hz, 3H, −CH3), 0.86–0.84 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.8 (C=O), 170.2 (C=O), 148.7 (Ar–C–NO2), 140.4 (Ar–C), 140.3 (2-N=CH), 137.4 (Ar–C), 134.6 (2Ar–C), 132.6 (Ar–CH), 129.8 (Ar–CH), 129.6 (2Ar–CH), 129.4 (2Ar–CH), 128.6 (Ar–C), 127.4 (2Ar–CH), 127.0 (2Ar–CH), 124.3 (Ar–CH), 121.6 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.8 (−CH), 30.1 (−CH), 22.3 (2-CH3), 18.5 (−CH3). HRMS (ESI+): 551.2729 [M + Na]+ calcd for C29H31NaN5O5: 551.2736.

N′-4-(2-(2-(3-Hydroxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5c)

Yellow amorphous solid: Yield: 85%, 106 mg; mp: 166–167 °C; 1H NMR (600 MHz, CDCl3): δ 9.27 (s, 2H, 2-NH), 7.76 (br.s, 1H, –N=CH−), 7.56 (s, 1H, –N=CH−), 7.30 (d, J = 7.8 Hz, 2H, Ar–H), 7.23–7.21 (m, 3H, Ar–H), 7.15–7.12 (m, 2H, Ar–H), 7.09–7.07 (m, 2H, Ar–H), 7.04 (d, J = 8.4 Hz, 2H, Ar–H), 6.88–6.86 (m, 1H, Ar–H), 4.68–4.67 (m, 2H, −CH2), 3.68–3.65 (m, 1H, −CH), 2.37 (d, J = 7.2 Hz, 2H −CH2), 1.81–1.76 (m, 1H, −CH), 1.50 (d, J = 6.6 Hz, 3H, −CH3), 0.83 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.1 (C=O), 171.7 (C=O), 156.6 (Ar–C–OH), 148.0 (–N=CH), 143.5 (–N=CH), 140.3 (Ar–C), 138.2 (Ar–C), 137.3 (Ar–C), 134.5 (Ar–C), 129.9 (Ar–CH), 129.7 (2Ar–CH), 129.6 (2Ar–CH), 127.6 (2Ar–CH), 127.4 (2Ar–CH), 127.0 (Ar–C), 121.1 (Ar–CH), 118.4 (Ar–CH), 113.6 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.1 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.2 (−CH3). HRMS (ESI+): 502.2975 [M + H]+ calcd for C29H34N4O4: 502.2983.

N′-4-(2-(2-(2-Hydroxy-3-methoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5d)

Pale yellow amorphous solid: Yield: 90%, 119 mg; mp: 171–172 °C; 1H NMR (600 MHz, CDCl3): δ 10.79 (br.s, 1H, −OH), 9.90 (s, 1H, –NH), 9.35 (s, 1H, –NH), 8.32 (s, 2H, 2-N=CH−), 7.28 (d, J = 7.8 Hz, 2H, Ar–H), 7.22 (d, J = 8.4 Hz, 1H, Ar–H), 7.13 (d, J = 7.8 Hz, 2H, Ar–H), 7.05 (d, J = 7.8 Hz, 2H, Ar–H), 6.91–6.90 (m, 1H, Ar–H), 6.84–6.83 (m, 2H, Ar–H), 6.78 (d, J = 7.2 Hz, 1H, Ar–H), 4.36–4.33 (m, 2H, −CH2), 3.90 (s, 3H, –OCH3), 3.65–3.61 (m, 1H, −CH), 2.38 (d, J = 7.2 Hz, 2H −CH2), 1.85–1.76 (m, 1H, −CH), 1.50 (d, J = 7.2 Hz, 3H, −CH3), 0.84 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.6 (C=O), 170.3 (C=O), 148.2 (Ar–C–OH), 147.1 (Ar–C–OCH3), 150.7 (–N=CH), 146.3 (–N=CH), 141.3 (Ar–C), 140.6 (Ar–C), 137.2 (Ar–C), 129.9 (2Ar–CH), 129.5 (2Ar–CH), 127.4 (2Ar–CH), 127.3 (2Ar–CH), 124.5 (Ar–C), 122.3 (Ar–CH), 119.5 (Ar–CH), 119.1 (Ar–C), 113.6 (Ar–CH), 69.0 (−CH2), 56.2 (−OCH3), 45.0 (−CH2), 41.8 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.4 (−CH3). HRMS (ESI+): 530.3537 [M + H]+ calcd for C30H34N4O5: 530.3541.

N′-(4-(2-(2-(2,4-Dihydroxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5e)

Light brown amorphous solid: Yield: 90%, 116 mg; mp: 191–193 °C; 1H NMR (600 MHz, CDCl3): δ 10.08 (br.s, 1H, −OH), 9.41 (br.s, 2H, 2-NH), 8.76 (br.s, 1H, –N=CH−), 7.99 (br.s, 1H, –N=CH−), 7.67 (s, 1H, Ar–H), 7.23–7.20 (m, 4H, Ar–H), 7.09–7.04 (m, 4H, Ar–H), 6.76 (br.s, 1H, Ar–H), 6.25 (br.s, 1H, Ar–H), 4.74 (br.s, 2H, −CH2), 3.75 (br.s, 1H, −CH), 2.43 (d, J = 7.2 Hz, 2H −CH2), 1.81–1.76 (m, 1H, −CH), 1.59 (d, J = 7.2 Hz, 3H, −CH3), 0.87 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.8 (C=O), 171.7 (C=O), 162.5 (Ar–C–OH), 162.2 (Ar–C–OH), 141.4 (Ar–C), 140.3 (2-N=CH), 137.4 (Ar–C), 134.6 (Ar–C), 133.8 (Ar–CH), 129.7 (2Ar–CH), 129.6 (2Ar–CH), 128.6 (Ar–C), 127.4 (2Ar–CH), 127.0 (2Ar–CH), 111.1 (Ar–C), 108.6 (Ar–CH), 103.7 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.8 (−CH), 30.2 (−CH), 22.3 (2-CH3), 18.1 (−CH3). HRMS (ESI+): 535.2598 [M + NH4]+ calcd for C29H36N5O5: 535.2676.

N′-(4-(2-(2-(2,4-Dimethoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5f)

Ash white amorphous solid: Yield: 88%, 117 mg; mp: 156–158 °C; 1H NMR (600 MHz, CDCl3): δ 9.90 (s, 1H, –NH), 9.35 (s, 1H, –NH), 8.40 (s, 1H, –N=CH−), 7.92 (s, 1H, –N=CH−), 7.78 (d, J = 8.4 Hz, 1H, Ar–H), 7.30 (d, J = 7.8 Hz, 2H, Ar–H), 7.11 (d, J = 8.4 Hz, 1H, Ar–H), 7.04 (d, J = 7.8 Hz, 3H, Ar–H), 6.53–6.52 (m, 2H, Ar–H), 6.40–6.38 (m, 2H, Ar–H), 4.77 (br.s, 2H, −CH2), 4.67–4.64 (m, 1H, −CH), 3.82 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.82–1.78 (m, 1H, −CH), 1.50 (d, J = 7.2 Hz, 3H, −CH3), 0.88–0.84 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.9 (C=O), 171.7 (C=O), 162.6 (Ar–C–OCH3), 159.2 (Ar–C–OCH3), 141.1 (Ar–C), 140.1 (–N=CH), 139.0 (–N=CH, Ar–C), 138.7 (Ar–C), 133.0 (Ar–C), 130.8 (Ar–C), 129.6 (2Ar–CH), 129.2 (2Ar–CH), 127.6 (2Ar–CH), 127.3 (2Ar–CH), 115.3 (Ar–C), 105.8 (Ar–CH), 98.0 (Ar–CH), 69.0 (−CH2), 55.5 (2-OCH3), 45.0 (−CH2), 41.3 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.3 (−CH3). HRMS (ESI+): 545.2764 [M + H]+ calcd for C31H37N4O5: 545.2724.

2-(4-Isobutyl phenyl)-N′-(4-(2-oxo-2-(2-(−3,4,5-trimethoxybenzylidene)hydrazinyl)ethoxy)benzylidene)propanehydrazide (5g)

Gray amorphous solid: Yield: 92%, 132 mg; mp: 195–196 °C; 1H NMR (600 MHz, CDCl3): δ 9.41 (br.s, 2H, 2-NH), 8.77 (s, 1H, –N=CH), 8.30 (br.s, 1H, –N=CH−), 7.95 (br.s, 1H, Ar–H), 7.52 (s, 1H, Ar–H), 7.28 (d, J = 8.4 Hz, 2H, Ar–H), 7.14–7.08 (m, 2H, Ar–H), 7.04 (d, J = 8.4 Hz, 2H, Ar–H), 6.82 (s, 2H, Ar–H), 4.72 (br.s, 2H, −CH2), 4.62–4.59 (m, 1H, −CH), 3.89 (s, 6H, –OCH3), 3.83 (s, 3H, –OCH3), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.84–1.76 (m, 1H, −CH), 1.51 (d, J = 7.2 Hz, 3H, −CH3), 0.89–0.84 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.1 (C=O), 171.7 (C=O), 153.5 (2Ar–C–OCH3), 142.5 (2-N=CH), 141.1 (Ar–C), 140.2 (Ar–C–OCH3), 139.8 (Ar–C), 138.7 (Ar–C), 129.9 (2Ar–CH), 129.2 (2Ar–CH), 127.4 (2Ar–CH), 127.0 (2Ar–CH), 128.6 (Ar–C), 128.0 (Ar–C), 104.1 (2Ar–CH), 69.0 (−CH2), 61.0 (−OCH3), 56.2 (2-OCH3), 45.0 (−CH2), 41.8 (−CH), 30.2 (−CH), 22.4 (2-CH3), 18.4 (−CH3). HRMS (ESI+): 593.3078 [M + NH4]+ calcd for C32H42N5O6: 593.3086.

N′-(4-(2-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5h)

Off white amorphous solid: Yield: 80%, 110 mg; mp: 168–169 °C; 1H NMR (600 MHz, CDCl3): δ 9.00 (s, 2H, 2-NH), 8.55 (s, 1H, –N=CH−), 8.29 (s, 1H, –N=CH−), 7.97 (s, 1H, Ar–H), 7.82 (d, J = 9.0 Hz, 1H, Ar–H), 7.36 (s, 1H, Ar–H), 7.31 (br.s, 1H, Ar–H), 7.26–7.19 (m, 4H, Ar–H), 7.13 (d, J = 7.8 Hz, 1H, Ar–H), 7.05 (d, J = 8.4 Hz, 2H, Ar–H), 4.60–4.56 (m, 2H, −CH2), 3.64–3.62 (m, 1H, −CH), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.82–1.76 (m, 1H, −CH), 1.51 (d, J = 7.2 Hz, 3H, −CH3), 0.84 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.2 (C=O), 170.6 (C=O), 142.6 (–N=CH), 141.3 (Ar–C), 140.3 (Ar–C), 138.4 (–N=CH), 137.3 (Ar–C), 130.5 (Ar–C–Cl), 130.3 (Ar–C), 130.2 (2Ar–CH), 129.9 (Ar–CH), 129.7 (Ar–CH), 129.3 (2Ar–CH), 128.6 (Ar–C–Cl), 128.0 (Ar–C), 127.8 (2Ar–CH), 127.6 (2Ar–CH), 127.4 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.6 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.5 (−CH3). HRMS (ESI+): 522.5970 [M + H]+ calcd for C29H30Cl2N4O3: 522.5982.

N′-(4-(2-(2-(3,5-Dibromo-4-hydroxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5i)

Brownish amorphous solid: Yield: 92%, 151 mg; mp: 138–140 °C; 1H NMR (600 MHz, CDCl3): δ 9.54 (s, 1H, −OH), 9.27 (br.s, 2H, 2-NH), 7.97 (s, 2H, 2-N=CH), 7.72 (br.s, 1H, Ar–H), 7.65 (s, 2H, Ar–H), 7.51 (s, 1H, Ar–H), 7.26 (d, J = 8.4 Hz, 2H, Ar–H), 7.23–7.21 (m, 1H, Ar–H), 7.14–7.09 (m, 1H, Ar–H), 7.06 (d, J = 7.8 Hz, 2H, Ar–H), 4.59–4.56 (m, 2H, −CH2), 3.64 (br.s, 1H, −CH), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.59–1.55 (m, 1H, −CH), 1.50 (d, J = 7.2 Hz, 3H, −CH3), 0.83 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.8 (C=O), 170.6 (C=O), 150.6 (Ar–C–OH), 144.5 (Ar–C), 141.3 (Ar–C), 140.3 (–N=CH), 139.9 (–N=CH), 137.3 (Ar–C), 131.0 (Ar–C), 130.5 (2Ar–CH), 129.9 (2Ar–CH), 129.6 (2Ar–CH), 129.3 (2Ar–CH), 127.4 (2Ar–CH), 127.0 (Ar–C), 110.3 (2Ar–C), 69.0 (−CH2), 45.0 (−CH2), 41.5 (−CH), 30.2 (−CH), 22.4 (2-CH3), 18.4 (−CH3). HRMS (ESI+): 657.4634 [M + H]+ calcd for C29H30Br2N4O4: 657.4650.

N′-(4-(2-(2-(4-Bromo-2-fluorobenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5j)

Orange amorphous solid: Yield: 92%, 133 mg; mp: 162–164 °C; 1H NMR (600 MHz, CDCl3): δ 8.89 (s, 2H, 2-NH), 8.46 (br.s, 1H, –N=CH), 8.12 (br.s, 1H, –N=CH), 7.89 (br.s, 1H, Ar–H), 7.78 (s, 1H, Ar–H), 7.71 (t, J = 7.8 Hz, 1H, Ar–H), 7.32 (d, J = 8.4 Hz, 1H, Ar–H), 7.26–7.19 (m, 4H, Ar–H), 7.13 (d, J = 8.4 Hz, 1H, Ar–H), 7.05 (d, J = 7.8 Hz, 2H, Ar–H), 4.61–4.57 (m, 2H, −CH2), 3.65–3.61 (m, 1H, −CH), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.80–1.76 (m, 1H, −CH), 1.51 (d, J = 7.2 Hz, 3H, −CH3), 0.86–0.82 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.3 (C=O), 170.6 (C=O), 161.6 (Ar–C–F), 159.9 (Ar–C), 140.3 (–N=CH), 139.4 (Ar–C), 135.1 (–N=CH, Ar–C), 130.0 (Ar–CH), 129.6 (2Ar–CH), 129.3 (2Ar–CH), 128.2 (Ar–C–Br), 128.0 (2Ar–CH), 127.4 (2Ar–CH), 127.3 (Ar–C), 127.0 (Ar–CH), 120.8 (Ar–C), 119.6 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.6 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.5 (−CH3). HRMS (ESI+): 598.1829 [M + NH4]+ calcd for C29H34BrFN5O3: 599.3258.

N′-(4-(2-(2-(2-Hydroxynaphthalen-1-yl)methylene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5k)

Yellow amorphous solid: Yield: 85%, 116 mg; mp: 159–161 °C; 1H NMR (600 MHz, CDCl3): δ 11.17 (br.s, 1H, −OH), 9.59 (br.s, 1H, –NH), 9.15 (s, 1H, –NH), 8.73 (s, 1H, –N=CH), 8.57 (br.s, 1H, –N=CH), 7.90 (d, J = 8.4 Hz, 1H, Ar–H), 7.85 (d, J = 8.4 Hz, 1H, Ar–H), 7.77–7.70 (m, 4H, Ar–H), 7.48 (t, J = 7.8 Hz, 1H, Ar–H), 7.41 (t, J = 7.2 Hz, 1H, Ar–H), 7.27–7.23 (m, 4H, Ar–H), 7.17 (d, J = 8.4 Hz, 2H, Ar–H), 4.37–4.34 (m, 2H, −CH2), 3.70 (br.s, 1H, −CH), 2.39 (d, J = 7.2 Hz, 2H −CH2), 1.85–1.76 (m, 1H, −CH), 1.56 (d, J = 6.6 Hz, 3H, −CH3), 0.83 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.2 (C=O), 170.2 (C=O, Ar–C–OH), 159.0 (Ar–C), 147.2 (–N=CH), 143.7 (–N=CH), 140.6 (Ar–C), 137.5 (Ar–C), 133.1 (Ar–CH), 132.0 (Ar–C), 130.0 (2Ar–CH), 129.6 (2Ar–CH), 129.5 (Ar–C), 127.7 (Ar–CH), 127.5 (2Ar–CH), 127.2 (2Ar–CH), 127.0 (Ar–CH, Ar–C), 123.4 (Ar–CH), 119.8 (Ar–CH), 118.6 (Ar–CH), 107.8 (Ar–C), 69.0 (−CH2), 45.0 (−CH2), 42.2 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.5 (−CH3). HRMS (ESI+): 569.2957 [M + NH4]+ calcd for C33H38N5O4: 569.1817.

N′-(4-(2-(2-(3,4-Dimethoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5l)

Ash white amorphous solid: Yield: 80%, 109 mg; mp: 148–150 °C; 1H NMR (600 MHz, CDCl3): δ 9.82 (s, 1H, –NH), 8.87 (s, 1H, –NH), 8.41 (br.s, 1H, –N=CH), 7.89 (s, 1H, –N=CH), 7.55 (s, 1H, Ar–H), 7.38 (s, 1H, Ar–H), 7.29 (d, J = 7.8 Hz, 2H, Ar–H), 7.24–7.22 (m, 2H, Ar–H), 7.11–7.07 (m, 2H, Ar–H), 6.98–6.93 (m, 2H, Ar–H), 6.83 (d, J = 8.4 Hz, 1H, Ar–H), 4.67–4.60 (m, 1H, −CH), 4.00 (s, 2H, −CH2), 3.88 (s, 6H, 2-OCH3), 2.37 (d, J = 6.6 Hz, 2H −CH2), 1.83–1.75 (m, 1H, −CH), 1.51 (d, J = 6.6 Hz, 3H, −CH3), 0.83 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.1 (C=O), 171.7 (C=O), 150.9 (Ar–C–OCH3), 149.6 (Ar–C–OCH3), 148.3 (Ar–C), 142.9 (2-N=CH), 140.2 (Ar–C), 138.7 (Ar–C), 130.1 (Ar–C), 129.8 (2Ar–CH), 129.2 (2Ar–CH), 127.4 (4Ar–CH), 126.2 (Ar–C), 121.8 (Ar–CH), 110.7 (Ar–CH), 108.3 (Ar–CH), 69.0 (−CH2), 56.0 (2-OCH3), 45.0 (−CH2), 41.6 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.5 (−CH3). HRMS (ESI+): 546.2798 [M + H]+ calcd for C31H37N4O5: 547.2941.

N′-(4-(2-(2-(2-Hydroxy-4-methoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5m)

Brownish amorphous solid: Yield: 88%, 116 mg; mp: 169–171 °C; 1H NMR (600 MHz, CDCl3): δ 11.08 (br.s, 1H, −OH), 9.90 (s, 1H, –NH), 9.36 (s, 1H, –NH), 8.37 (br.s, 1H, –N=CH), 8.31 (br.s, 1H, –N=CH), 7.83 (s, 1H, Ar–H), 7.27 (d, J = 7.8 Hz, 2H, Ar–H), 7.16–7.08 (m, 4H, Ar–H), 7.05 (d, J = 7.8 Hz, 2H, Ar–H), 6.95–6.94 (m, 2H, Ar–H), 4.68 (s, 2H, −CH2), 4.36–4.32 (m, 1H, −CH), 3.90 (s, 3H, –OCH3), 2.37 (d, J = 7.2 Hz, 2H −CH2), 1.83–1.75 (m, 1H, −CH), 1.50 (d, J = 6.6 Hz, 3H, −CH3), 0.83 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.6 (C=O), 164.8 (C=O), 150.7 (Ar–C–OH), 149.7 (Ar–C–OCH3), 148.3 (Ar–C), 146.2 (–N=CH), 141.3 (Ar–C), 140.6 (–N=CH), 137.3 (Ar–C), 129.9 (Ar–CH), 129.6 (Ar–CH), 129.5 (2Ar–CH), 127.3 (2Ar–CH), 127.0 (Ar–C), 124.5 (Ar–CH), 124.1 (Ar–CH), 122.3 (Ar–CH), 119.0 (Ar–C), 117.9 (Ar–CH), 115.2 (Ar–CH), 69.0 (−CH2), 56.2 (−OCH3), 45.0 (−CH2), 41.8 (−CH), 30.1 (−CH), 22.4 (2-CH3), 19.0 (−CH3). HRMS (ESI+): 530.0492 [M + H]+ calcd for C30H34N4O5: 530.0505.

N′-(4-(2-(2-(3-Hydroxy-4-methoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5n)

White amorphous solid: Yield: 78%, 103 mg; mp: 110–113 °C; 1H NMR (600 MHz, CDCl3): δ 9.81 (s, 2H, 2-NH), 8.87 (s, 1H, –N=CH), 7.54 (s, 1H, –N=CH), 7.41 (s, 2H, Ar–H), 7.30–7.29 (m, 3H, Ar–H), 7.15–7.07 (m, 2H, Ar–H), 7.04 (d, J = 7.8 Hz, 3H, Ar–H), 6.94 (d, J = 8.4 Hz, 1H, Ar–H), 6.82 (d, J = 8.4 Hz, 1H, Ar–H), 4.77 (br.s, 2H, −CH2), 4.68–4.65 (m, 1H, −CH), 3.90 (s, 3H, –OCH3), 2.38 (d, J = 7.2 Hz, 2H −CH2), 1.82–1.75 (m, 1H, −CH), 1.50 (d, J = 7.2 Hz, 3H, −CH3), 0.83 (d, J = 6.0 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.4 (C=O), 171.7 (C=O), 152.1 (Ar–C–OCH3), 148.3 (Ar–C–OH), 146.1 (Ar–C), 145.9 (–N=CH), 143.0 (–N=CH), 140.1 (Ar–C), 138.4 (Ar–C), 130.4 (Ar–C), 129.8 (Ar–CH), 129.6 (Ar–CH), 129.2 (2Ar–CH), 127.6 (2Ar–CH), 127.5 (Ar–CH), 127.0 (Ar–CH), 126.6 (Ar–C), 120.7 (Ar–CH), 114.1 (Ar–CH), 112.0 (Ar–CH), 69.0 (−CH2), 56.0 (−OCH3), 45.0 (−CH2), 41.1 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.2 (−CH3). HRMS (ESI+): 531.2607 [M + H]+ calcd for C30H35N4O5: 531.2659.

N′-(4-(2-(2-(4-(Diethylamino)benzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5o)

Pale yellow amorphous solid: Yield: 78%, 108 mg; mp: 151–153 °C; 1H NMR (600 MHz, CDCl3): δ 9.41 (br.s, 2H, 2-NH), 8.46 (s, 2H, 2-N=CH), 7.68 (d, J = 8.4 Hz, 1H, Ar–H), 7.58–7.43 (m, 4H, Ar–H), 7.31–7.29 (m, 2H, Ar–H), 7.10–7.06 (m, 1H, Ar–H), 7.04 (d, J = 7.8 Hz, 2H, Ar–H), 6.65 (d, J = 9.0 Hz, 2H, Ar–H), 4.76 (s, 2H, −CH2), 4.67 (br.s, 1H, −CH), 3.42–3.37 (m, 4H, 2-CH2), 2.38 (d, J = 7.2 Hz, 2H −CH2), 1.81–1.76 (m, 1H, −CH), 1.51 (d, J = 7.2 Hz, 3H, −CH3), 1.20–1.12 (m, 6H, 2-CH3), 0.84–0.83 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.8 (C=O), 171.7 (C=O), 148.8 (Ar–C–N(CH2CH3)2), 141.0 (Ar–C), 140.3 (2-N=CH), 140.1 (Ar–C), 138.7 (Ar–C), 129.8 (2Ar–CH), 129.2 (2Ar–CH), 128.7 (2Ar–CH), 127.6 (2Ar–CH), 127.5 (2Ar–CH), 127.0 (Ar–C), 111.2 (Ar–C), 110.6 (2Ar–CH), 69.0 (−CH2), 45.1 (2-CH2), 44.7 (−CH2), 41.1 (−CH), 30.2 (−CH), 22.4 (2-CH3), 18.2 (−CH3), 12.5 (−2-CH3). HRMS (ESI+): 556.3288 [M + H]+ calcd for C33H42N5O3: 556.3292.

2-(4-Isobutyl phenyl)-N′-(4-(2-(2-(3-methoxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)propanehydrazide (5p)

White amorphous solid: Yield: 84%, 107 mg; mp: 132–134 °C; 1H NMR (600 MHz, CDCl3): δ 9.81 (s, 2H, 2-NH), 8.87 (s, 1H, –N=CH), 7.54 (s, 1H, –N=CH), 7.60 (s, 1H, Ar–H), 7.41–7.25 (m, 3H, Ar–H), 7.22 (br.s, 2H, Ar–H), 7.13–7.07 (m, 2H, Ar–H), 7.03 (d, J = 7.2 Hz, 2H, Ar–H), 6.91–6.90 (m, 2H, Ar–H), 4.68 (s, 2H, −CH2), 4.66 (br.s, 1H, −CH), 3.83 (s, 3H, –OCH3), 2.37 (d, J = 7.2 Hz, 2H −CH2), 1.83–1.75 (m, 1H, −CH), 1.51 (d, J = 6.6 Hz, 3H, −CH3), 0.83 (d, J = 6.0 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.5 (C=O), 170.7 (C=O), 159.8 (Ar–C–OCH3), 143.0 (2-N=CH), 141.1 (Ar–C), 140.0 (Ar–C), 138.5 (Ar–C), 135.2 (Ar–C), 130.0 (2Ar–CH), 129.9 (Ar–C), 129.7 (Ar–CH), 129.2 (2Ar–CH), 127.5 (2Ar–CH), 127.1 (2Ar–CH), 120.3 (Ar–CH), 116.2 (Ar–CH), 111.2 (Ar–CH), 68.3 (−CH2), 55.3 (−OCH3), 45.0 (−CH2), 41.4 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.3 (−CH3). HRMS (ESI+): 515.2658 [M + H]+ calcd for C30H35N4O4: 515.2745.

N′-(4-(2-(2-(4-Fluorobenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5q)

Off white amorphous solid: Yield: 92%, 119 mg; mp: 185–187 °C; 1H NMR (600 MHz, CDCl3): δ 9.41 (br.s, 2H, 2-NH), 8.84 (s, 2H, 2-N=CH), 7.59–7.57 (m, 4H, Ar–H), 7.29 (d, J = 7.8 Hz, 4H, Ar–H), 7.23–7.14 (m, 4H, Ar–H), 4.65–4.62 (m, 2H, −CH2), 3.69–3.62 (m, 1H, −CH), 2.39 (d, J = 6.6 Hz, 2H, −CH2), 1.83–1.76 (m, 1H, −CH), 1.52 (d, J = 7.2 Hz, 3H, −CH3), 0.88–0.85 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.3 (C=O), 171.7 (C=O), 162.4 (Ar–C–F), 147.3 (Ar–C), 141.7 (2-N=CH), 140.2 (Ar–C), 138.4 (Ar–C), 130.0 (Ar–C), 129.2 (4Ar–CH), 128.9 (2Ar–CH), 127.5 (2Ar–CH), 127.2 (2Ar–CH), 127.0 (Ar–C), 115.7 (2Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.4 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.3 (−CH3). HRMS (ESI+): 520.2724 [M + NH4]+ calcd for C29H35FN5O3: 520.3336.

N′-(4-(2-(2-(2-Hydroxybenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)-2-(4-isobutyl phenyl)propanehydrazide (5r)

Off white amorphous solid: Yield: 80%, 109 mg; mp: 154–155 °C; 1H NMR (600 MHz, CDCl3): δ 10.98 (s, 1H, −OH), 9.93 (br.s, 1H, –NH), 9.87 (s, 1H, –NH), 8.42 (br.s, 1H, –N=CH), 8.22 (br.s, 1H, –N=CH), 7.79 (s, 1H, Ar–H), 7.54–7.48 (m, 2H, Ar–H), 7.36–7.20 (m, 4H, Ar–H), 7.15–7.12 (m, 4H, Ar–H), 6.93–6.86 (m, 1H, Ar–H), 4.30–4.27 (m, 2H, −CH2), 3.64 (br.s, 1H, −CH), 2.38 (d, J = 6.6 Hz, 2H −CH2), 1.84–1.76 (m, 1H, −CH), 1.50 (d, J = 7.2 Hz, 3H, −CH3), 0.83 (d, J = 7.2 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 175.3 (C=O), 164.7 (C=O), 161.6 (Ar–C), 158.5 (Ar–C–OH), 146.9 (–N=CH), 140.6 (Ar–C), 137.5 (Ar–C), 137.0 (–N=CH), 133.7 (Ar–CH), 130.0 (2Ar–CH), 129.6 (2Ar–CH), 127.4 (2Ar–CH), 127.1 (3Ar–CH), 120.7 (Ar–C), 119.8 (Ar–CH), 119.3 (Ar–C), 117.6 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 42.2 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.4 (−CH3). HRMS (ESI+): 520.2801 [M + NH4]+ calcd for C31H40N5O5: 520.3327.

2-(4-Isobutyl phenyl)-N′-(4-(2-(2-(4-isopropylbenzylidene)hydrazinyl)-2-oxoethoxy)benzylidene)propanehydrazide (5s)

White amorphous solid: Yield: 80%, 105 mg; mp: 164–166 °C; 1H NMR (600 MHz, CDCl3): δ 9.59 (br.s, 1H, –NH), 9.15 (s, 1H, –NH), 8.91 (s, 2H, 2-N=CH), 7.61 (s, 1H, Ar–H), 7.58–7.55 (m, 1H, Ar–H), 7.53 (d, J = 8.4 Hz, 2H, Ar–H), 7.31 (d, J = 7.8 Hz, 2H, Ar–H), 7.27–7.23 (m, 2H, Ar–H), 7.18 (d, J = 7.8 Hz, 1H, Ar–H), 7.12 (d, J = 7.8 Hz, 1H, Ar–H), 7.05 (d, J = 8.4 Hz, 2H, Ar–H), 5.22–5.17 (m, 1H, −CH), 4.77 (s, 2H, −CH2), 4.70–4.66 (m, 1H, −CH), 2.38 (d, J = 6.6 Hz, 2H −CH2), 1.83–1.75 (m, 1H, −CH), 1.52 (d, J = 7.2 Hz, 3H, −CH3), 1.26–1.21 (m, 6H, 2-CH3), 0.88–0.81 (m, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.4 (C=O), 171.7 (C=O), 151.6 (Ar–C–CH(CH3)2), 151.3 (–N=CH), 147.9 (Ar–C), 143.1 (–N=CH), 140.2 (Ar–C), 138.5 (Ar–C), 130.0 (Ar–C), 129.2 (2Ar–CH), 128.6 (2Ar–CH), 128.0 (Ar–C), 127.6 (2Ar–CH), 127.4 (2Ar–CH), 127.1 (2Ar–CH), 126.9 (2Ar–CH), 69.0 (−CH2), 45.1 (−CH2), 41.2 (−CH), 34.1 (−CH), 30.2 (−CH), 23.8 (2-CH3), 22.4 (2-CH3), 18.2 (−CH3). HRMS (ESI+): 527.3022 [M + H]+ calcd for C32H39N4O3: 527.3038.

2-(4-Isobutyl phenyl)-N′-(4-(2-(2-(naphthalen-1-ylmethylene)hydrazinyl)-2-oxoethoxy)benzylidene)propanehydrazide (5t)

Yellowish amorphous solid: Yield: 85%, 113 mg; mp: 141–142 °C; 1H NMR (600 MHz, CDCl3): δ 10.38 (s, 1H, –NH), 9.30 (s, 1H, –NH), 8.59 (d, J = 8.4 Hz, 2H, Ar–H), 8.26 (s, 2H, 2-N=CH), 7.98–7.91 (m, 1H, Ar–H), 7.89–7.79 (m, 5H, Ar–H), 7.61–7.54 (m, 3H, Ar–H), 7.36 (d, J = 7.8 Hz, 2H, Ar–H), 7.07 (d, J = 7.8 Hz, 2H, Ar–H), 4.77–4.72 (m, 1H, −CH), 4.68 (s, 2H, −CH2), 2.38 (d, J = 7.2 Hz, 2H −CH2), 1.80–1.75 (m, 1H, −CH), 1.53 (d, J = 6.6 Hz, 3H, −CH3), 0.86 (d, J = 6.6 Hz, 6H, 2-CH3). 13C NMR (150 MHz, CDCl3): δ 176.6 (C=O), 162.0 (C=O), 143.2 (2-N=CH), 140.3 (Ar–C), 138.5 (Ar–C), 136.7 (Ar–C), 132.0 (Ar–C), 131.9 (Ar–C), 130.7 (Ar–CH), 129.6 (2Ar–CH), 129.3 (2Ar–CH), 128.8 (Ar–C), 128.2 (Ar–CH), 128.1 (2Ar–CH), 127.5 (2Ar–CH), 127.3 (Ar–CH), 126.3 (Ar–CH, Ar–C), 126.2 (Ar–CH), 125.3 (Ar–CH), 124.2 (Ar–CH), 69.0 (−CH2), 45.0 (−CH2), 41.6 (−CH), 30.1 (−CH), 22.4 (2-CH3), 18.7 (−CH3). HRMS (ESI+): 535.2709 [M + H]+ calcd for C33H35N4O3: 535.2798.

Anticancer Activity

Cell Lines and Culture Medium

U87-malignant glioma (ATCC-HTB-14) and human embryonic kidney HEK293 cell lines were obtained from the cell culture bank of the Atta-Ur-Rahman School of Applied Biosciences, National University of Science and Technology (NUST), Islamabad, Pakistan. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin solutions in a humidified incubator set at 37 °C with 5% carbon dioxide.

MTT Assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay involves the estimation of the metabolic activity of the living cells and is the most common method used for measuring cytotoxicity of any substance. Cytotoxicity is expressed as the concentration of the synthetic compounds that inhibited the growth of cells by 50% (IC50).46 The principle behind this assay is the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) reagent to insoluble formazan crystals by viable cells, and hence, cellular viability is measured. In the current study, MTT assay was performed to check the cytotoxicity of 5a–t compounds against the human cancerous cell line U87-malignant glioma and human embryonic kidney HEK293 as a control by adding them at a density of 1 × 103 cells per well in the 96-well culture plate in a humidified incubator having 5% CO2 at 37 °C. Twenty-four hours post plating when the cells were adhered, 100 μL of varying concentrations of test compounds (4, 5a–t) ranging from 10 to 150 μM (10, 40, 80, 100, and 150 μM) were added to the cells and incubated for 48 h at 37 °C in an incubator. After 24 h of addition of the drugs, cytotoxicity was checked by adding 50 μM. MTT solution prepared in 15 μL of phosphate-buffered saline (PBS) (BIO BASIC Canada INC.) to each well and incubated for 3 h. Furthermore, the supernatant was discarded, and then, 100 μL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to all of the wells to dissolve formazan crystals and incubated again for 30 min. The optical density was measured at a wavelength of 550 nm using a microplate reader (BIO-RAD PR4100). From the obtained data of absorbance, % cell viability was calculated using the following equation46,47

graphic file with name ao3c07216_m001.jpg

Molecular Docking

Molecular docking is an important tool for investigating interactions between an inhibitor molecule and a protein target.48 The crystal structure of casein kinase-2 (CK2) (PDB id: 3PE1) was downloaded from protein data bank (http://www.rcsb.org). The structure of the synthesized compounds was built in MOE and energy minimized using the default parameter of MOE (www.chemcomp.com). The active site residues of CK2 binding pockets were selected from the previous literature43 and the synthesized compounds were docked with the active residues of CK2 according to the most standard parameter, i.e., Placement Triangle Matcher rescoring: London dG. For each compound, 10 conformations were generated. The top-ranked conformation of each compound was used for further analysis.

ADMET Study

For the drug likeliness study, the SwissADME server was used to estimate the pharmacokinetic properties of the product compounds (5a–t), which were evaluated by uploading their structures in SMILE format to the SwissADME Web site (http://www.swissadme.ch/).49 Web tool characterizing small molecules according to physicochemical parameters including MW, TPSA, HB donor, HB acceptor, rotational bond number, Log P, Log S, and Lipinski filter violation of the SwissADME program was used to investigate the drug similarity of the synthesized molecules.

Results and Discussion

Chemistry

In the present research work, efforts were made to synthesize different derivatives based on the commercially available drug ibuprofen successfully synthesized via many step reactions in decent yields. In the first step, the acid of ibuprofen was refluxed with the coupling reagent 1,1-carbonyldiimidazole (CDI) in the existence of triethyl amine as a base in tetrahydrofuran (THF) solvent, and then hydrazine hydrate was added to the reaction mixture and further refluxed for 4–5 h. In the second step, the desired hydrazide was treated with esterified aldehyde in acetic acid as the catalyst for 3–4 h with constant stirring to get hydrazone-Schiff base derivative. The hydrazone-Schiff base was further reacting with excess of hydrazine hydrate in absolute ethanol to obtain the Schiff’s base hydrazide. In the last step, the Schiff base hydrazide was refluxed with different aromatic aldehydes in a catalytic amount of acetic acid in absolute ethanol to obtain bis-Schiff base derivatives of ibuprofen (Scheme 1). The synthesized derivatives were structurally deduced through various spectroscopic techniques including HR-ESI-MS, 1H NMR, and 13C NMR. Finally, we screened the obtained derivatives for their anticancer activity using a malignant glioma U87 cancerous cell line as well as a human embryonic kidney Hek293 cell line as a control.

Scheme 1. Synthesis of Ibuprofen-Based bis-Schiff Bases 5a–t.

Scheme 1

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The proton NMR spectrum of bis-Schiff bases of ibuprofen showed signals for methyl (−CH3), methylene (−CH2), methine (−CH), and –NH protons. In the downfield region of the spectrum, signals resonated in the region 9.90–8.63 were assigned to the –NH protons. Furthermore, signals appearing in the aromatic region 8.19–6.86 were due to the aromatic protons. In the spectrum, broad singlet signals in the region 4.70–4.65 were allotted to that of methylene protons, while in the upfield region of the spectrum, the methyl protons were seen at 0.80–0.89 ppm. Similar to this, the broad band 13C NMR spectrum of these product compounds showed signals for methyl, methylene, methine, and quaternary carbons, which were further solved by its distortionless enhancement by the polarization transfer (DEPT) spectrum. In the downfield region of the spectrum, signals resonated in the region 177.9–170.1 were assigned to carbonyl carbon atoms. On the other hand, signals appearing in the region 150.4–145.1 were due to the azomethine carbon atom. Moreover, signals of the methylene carbon atoms were seen in the 69.0–45.2 region, while the signals of methyl carbon atoms were seen at 24.8–18.2 ppm. The molar masses of these product derivatives were confirmed by its ESI-MS spectrum showing the molecular ion peaks. The detailed spectral interpretation of product compounds is present in the experimental section.

Anticancer Activity

The anticancer potential of ibuprofen-based bis-Schiff bases was screened against malignant glioma U87 cell line and human embryonic kidney HEK293 cell line. The findings are presented in Table 2. A dose-dependent anticancer response was noticed with increasing concentration of the test compounds, and sharp reduction in the viability percentage of the cells was noticed. However, at higher drug doses, the % survival decrease of the cells was relatively low. Different concentrations (10, 40, 80, 100, and 150 μM) of the synthesized ibuprofen bis-Schiff bases (5a–t) and compound 4 were used to investigate the growth inhibition of cancer cells in the human brain cancer cell line (malignant glioma U87). Simultaneously, human normal embryonic kidney cells (HEK293) were also used in the experiment as a positive control. To ascertain the reduction in cancer cell viability brought on by cytotoxic drugs, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was utilized. For human malignant glioma U87, the IC50 values, and % cell viability of the compounds (5a–t) are presented in Table 2. Using GraphPad Prism 8.0.2, the dose–response and IC50 values were determined. The results of the MTT assay revealed that all compounds showed excellent to less antiproliferative activity in malignant glioma U87 cells with IC50 values in the range of 5.75 ± 0.43 to 150.45 ± 0.20 μM. Among the series, compound 5e (IC50 = 5.75 ± 0.43 μM) is the most potent compound, while compounds 5f (IC50 = 24.17 ± 0.46 μM), 5b (IC50 = 35.73 ± 0.09 μM), 5a (IC50 = 36.7 ± 0.14 μM), 5n (IC50 = 40.18 ± 0.07 μM), 5r (IC50 = 43.66 ± 0.04 μM), 5s (IC50 = 43.73 ± 0.11 μM), 5g (IC50 = 39.94 ± 0.16 μM), 5q (IC50 = 46.0 ± 0.08 μM), 5i (IC50 = 38.32 ± 0.10 μM), and 5j (IC50 = 43.71 ± 0.07 μM) exhibited good inhibitory potency toward the brain cancer cell line (malignant glioma U87). Human embryonic kidney HEK293 cells were also exposed to the compounds at varying concentrations (10, 40, 80, 100, and 150 μM) in a manner similar to that of the cancer cells in order to examine the selective cytotoxic effects of compounds for malignant cells as compared to nonmalignant cells. The findings suggest that these cells (HEK293) are less vulnerable to the activities of compounds, particularly 5e, 5f, and 5b, which increases the death of brain cancer cells. It is revealed that the aggressive brain cancer cell line (malignant glioma U87) responded more positively to the majority of the chemicals with higher cytotoxicity. The lower cytotoxicity of compounds (5e, 5f, and 5b) in noncancerous cells suggests that these novel compounds will provide promising treatment/therapy for patients with brain cancer (Figure 3). Similarly, three compounds, i.e., 5k, 5c, and 5l showed significant activity against the malignant glioma U87 having IC50 values of 52.81 ± 0.06, 62.89 ± 0.15, and 72.83 ± 0.06 μM, respectively. Furthermore, the remaining seven derivatives (5t, 5p, 5h, 5d, 5o, and 5m) were found moderate to less active in the range of IC50 values of 86.74 ± 0.26 to 150.45 ± 0.20 μM.

Table 2. % Cell Viability (IC50) of the Active Analogues of Compounds (5a–t) against Brain Cancer (U87) and Normal (HEK293) Cell Lines.

compds concentration % cell viability U87 % cell viability HEK293 IC50 (μM ± SEM U87)
4 10 72.48 29.06 59.9 ± 0.08
40 47.92 28.63
80 41.59 28.20
100 41.29 26.30
150 33.69 9.96
5a 10 89.95 49.34 36.70 ± 0.14
40 88.02 48.55
80 77.41 47.25
100 73.86 45.20
150 61.75 38.29
5b 10 80.51 111.70 35.73 ± 0.09
40 75.83 101.63
80 70.36 95.98
100 62.23 94.29
150 59.10 91.18
5c 10 119.53 58.63 62.89 ± 0.15
40 108.60 55.19
80 107.07 49.58
100 106.67 49.52
150 94.78 47.63
5d 10 55.95 64.08 122.46 ± 0.14
40 51.96 55.95
80 40.37 49.81
100 27.60 43.64
150 22.08 39.42
5e 10 43.42 69.42 5.75 ± 0.43
40 41.01 69.09
80 39.87 67.88
100 28.47 62.10
150 27.48 50.83
5f 10 54.09 102.53 24.17 ± 0.46
40 44.25 79.22
80 38.69 71.94
100 28.96 69.91
150 21.62 63.50
5g 10 56.40 86.66 39.94 ± 0.16
40 47.40 75.46
80 39.45 62.21
100 33.32 62.12
150 30.90 56.11
5h 10 50.20 84.13 120.07 ± 0.13
40 50.15 79.45
80 48.83 69.13
100 45.96 68.32
150 43.63 67.09
5i 10 40.32 67.56 38.32 ± 0.10
40 36.85 57.35
80 31.43 55.73
100 29.86 49.92
150 24.43 43.82
5j 10 186.64 44.54 43.71 ± 0.07
40 128.08 40.06
80 94.20 38.35
100 93.14 36.79
150 78.60 26.31
5k 10 121.23 42.15 52.81 ± 0.06
40 100.37 35.61
80 99.04 28.64
100 96.61 25.74
150 90.20 25.47
5l 10 96.97 30.48 72.83 ± 0.06
40 88.44 28.12
80 84.89 25.92
100 84.18 24.86
150 79.72 24.19
5m 10 141.99 26.18 150.45 ± 0.20
40 138.25 26.12
80 34.46 24.16
100 134.23 21.90
150 127.60 18.62
5n 10 164.68 38.13 40.18 ± 0.07
40 85.29 27.99
80 61.13 24.79
100 42.70 16.88
150 33.17 12.62
5o 10 146.33 49.04 119.73 ± 0.25
40 121.87 42.87
80 120.97 37.72
100 119.00 35.75
150 106.95 35.19
5p 10 150.92 49.89 90.68 ± 0.13
40 121.78 49.35
80 93.71 49.16
100 93.65 48.09
150 93.46 39.27
5q 10 182.99 27.52 46.0 ± 0.08
40 84.04 25.39
80 81.65 19.95
100 48.50 19.70
150 40.48 17.80
5r 10 109.42 39.02 43.66 ± 0.04
40 81.40 33.62
80 72.77 31.98
100 45.45 26.11
150 40.62 25.06
5s 10 145.03 58.72 43.73 ± 0.11
40 93.13 58.38
80 92.16 51.45
100 78.36 49.00
150 69.18 39.29
5t 10 179.1 68.74 86.74 ± 0.26
40 169.25 58.86
80 155.44 51.05
100 146.07 47.32
150 139.58 43.01
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Figure 3.

Figure 3

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Percentage cell viability of the most potent compounds (A) 5b, (B) 5e, and (C) 5f at increasing concentrations ranging from 10 to 150 μM on the U87-MG cell line and HEK293 Human embryonic kidney cell line using the in vitro MTT assay.

Structure–Activity Relationship Study

In order to clarify the structure–activity relationship (SAR), modifications in the position and type of attached substituents (R) on the benzene ring were investigated. Compound 5e in the series was the most potent having an IC50 value of 5.75 ± 0.43 μM; the potency of this compound could be due to the attachment of two electron-donating hydroxyl groups at ortho and para position of the benzene ring. Similarly, a slight decrease occurs in the activity of compound 5f (IC50 = 24.17 ± 0.46 μM) that might be due to the change of substituents to methoxy groups at the same position compared with compound 5e (Figure 4). Comparing compounds 5a with 5b, the high activity of compound 5b (IC50 = 35.73 ± 0.09 μM) could be due to the attachment of the nitro group at the meta position instead of compound 5a (IC50 = 36.7 ± 0.14 μM) having the same group at the para position of the benzene ring showing little low activity. Furthermore, by comparison of compounds 5n with 5m and 5r, the highest activity of compound 5n (IC50 = 40.18 ± 0.07 μM) may be due to the presence of the hydroxyl group at meta position and methoxy group at the para position of the benzene ring. Similarly, by replacing the methoxy groups instead of hydroxyl groups from the meta position may reduce the activity of compound 5r (IC50= 43.66 ± 0.04 μM). Changing the position of the hydroxyl substituent from meta to ortho is responsible for the decrease in the activity of compound 5m (IC50= 150.45 ± 0.20 μM). On the other hand, compounds 5g (IC50= 39.94 ± 0.16 μM) and 5i (IC50= 38.32 ± 0.10 μM) displayed almost same activity, while the greater activity of 5g could be due to the attachment of three electron-donating methoxy substituents at meta and para positions. However, a slight decline in the activity of compound 5i may be likely due to the replacement of two methoxy groups with bromine substituents at the meta position of the benzene ring. The higher inhibitory activity of 5j (IC50 = 43.71 ± 0.07 μM) than 5h (IC50 = 120.07 ± 0.13 μM) might be possibly due to the attachment of two electron-withdrawing fluorines at the ortho and bromine at the para position to the benzene ring by replacing fluorine and bromine groups with two chlorine substituents at the same positions, decreasing the activity.

Figure 4.

Figure 4

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Most active product compounds (5e, 5f, 5b, 5a, 5n, and 5s) in the synthetic series (5a–t).

Molecular Docking

The synthesized series, having 20 compounds (5a–t) docked with a three-dimensional crystal structure of human casein kinase-2 CK2 (PDB ID: 3PE1) active site residues of casein kinase-2, which include LEU45, LYS49, VAL53, GLU55, LYS68, VAL116, ASN118, HIS160, ASP175, and TRP176, were interacting residues. The top three compounds were selected according to S-scores and interactions; Table 2 showed S-scores and interaction residues of compounds with the CK2 enzyme. Among 20 docked compounds, 5b, 5f, and 5e showed the best docking scores and interaction with protein as compared to doxorubicin (reference). Figure 5 shows two-dimensional (2D) interactions of all compounds and three-dimensional (3D) interactions of active site residues of the CK2 protein with compounds.

Figure 5.

Figure 5

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Best docking scores and interaction of compounds (5b, 5f, and 5e) with protein as compared to doxorubicin.

Molecular Docking Studies

Among the series, compound 5b was found as one of the most active compounds with an S-score of −7.46. The carbon atom of compound 5b established one H-donor interaction with MET163, while the 6-ring of compound 5b formed two π–H contacts with LYS49 and VAL53. Similarly, compound 5f was found as the second most potent compound of the series with an S-score of −7.37. The carbon atom of compound 5f recognized a total of one H-bond donor and the 6-ring of compound 5f formed two π–H contacts with ASP175, LYS49, and TYR50, respectively. Based on the docking scores, compound 5e was found as the third potent compound of series with an S-score of −6.83. The carbon and oxygen atoms of compound 5e formed two H-bond donor interactions with GLU55 and ASN118, respectively. Similarly, the 6-ring of compound 5e formed two π–H interactions with LYS49 and VAL53. Table 3 shows the S-scores and interaction details of all of the synthetic 20 (5a–t) compounds of the series and reference. The 2D interactions of all of the compounds against the receptor are shown in Figure S1, while the 3D interactions of the most potent compounds along with reference compounds are shown in Figure 6.

Table 3. Comparison of Docking Scores and Interactions of the Produced Compounds with Reference Doxorubicin.

compound ID docking score (kcal/Mole) interacting residues bond type distance (Å)
doxorubicin –6.0721 HIS160 H-donor 3.5
HIS160 π–π 3.2
HIS160 π–π 3.6
5a –7.2614 LEU45 H-donor 3.32
LEU45 π–H 4.32
LYS49 π–H 4.02
5b –7.4617 MET163 H-donor 3.8
LYS49 π–H 4.2
VAL53 π–H 4.1
5c –7.1320 GLU55 H-donor 3.05
ASN118 H-acceptor 3.08
5d –7.0399 MET163 H-donor 3.83
LYS49 H-acceptor 3.21
SER 194 π–H 3.89
5e –6.8309 GLU55 H-donor 3.5
ASN118 H-donor 2.9
LYS49 π–H 4.0
VAL53 π–H 4.0
5f –7.3742 ASP175 H-donor 4.3
LYS49 π–H 6.5
TYR50 π–H 4.8
5g –7.6728 LYS49 π–H 4.09
VAL53 π–H 3.63
5h –6.7745 GLU55 H-donor 3.71
GLU55 H-donor 3.50
LEU45 π–H 4.17
5i –6.4936 GLU55 H-donor 3.39
ASN118 H-acceptor 2.98
PHE 121 π–π 3.72
5j –7.1744 LYS49 H-acceptor 3.41
LEU45 π–H 4.37
5k –7.5003 ASN118 H-acceptor 2.86
LYS49 π–H 4.61
5l –7.3675 HIS160 H-donor 3.32
VAL53 π–H 4.36
SER 194 π–H 3.54
ARG 195 π–H 4.47
5m –6.7229 LEU45 π–H 4.09
LYS49 π–H 4.27
5n –6.8956 LYS 44 H-acceptor 3.06
LYS49 π–H 4.01
5o –6.9995 GLY 46 π–H 4.40
LYS49 π–H 4.63
5p –7.4664 ASP 156 H-donor 3.22
LYS49 H-acceptor 3.38
LYS49 π–H 4.64
5q –6.0085 LEU45 H-donor 3.24
GLY 46 π–H 4.33
5r –7.0985 LYS 158 H-acceptor 3.37
ASN 161 H-acceptor 3.35
5s –7.0009 LYS49 H-acceptor 3.20
LYS49 Pi-cation 3.74
SER 194 π–H 3.98
5t –6.8773 ASN 117 H-acceptor 3.31
ASN118 H-acceptor 2.95
LEU45 π–H 4.12
LYS49 π–H 4.26
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Figure 6.

Figure 6

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3D interactions of the most potent compounds (5b, 5e, and 5f) along with reference.

ADMET Study

Drug likeliness is important in terms of a molecule’s potential to become a drug. SwissADME is a freely available web-based tool (http://www.swissadme.ch/) that estimates drug affinities by providing pharmacokinetic profiles of small molecules. SwissADME characterizes small molecules using Lipinski’s rule of five to determine the drug likeness of molecules (Figure 7). According to this rule, the molar mass should be <500, the number of H-bond acceptors <10, the number of H-bond donors <5, and Log P < 5 (or Mlog P < 4.15). Table S1 clearly shows that the compounds tested in the study comply with this rule but the molar mass of compounds is more than 500. The TPSA value is also within the acceptable range and the bioavailability score and synthetic availability values are also satisfactory.

Figure 7.

Figure 7

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Bioavailability radar of various bioactive drug-like molecules (5a–5t); the red areas in discern constitute every attribute of lipophilicity, solubility, molecular weight, and versatility using the SwissADME predictor.

Conclusions

In the current study, we have successfully synthesized 21 novel bis-Schiff base derivatives based on the commercially available drug ibuprofen in better yields. Structures of these derivatives were deduced through modern spectroscopic techniques including HR-ESI-MS, 1H NMR, and 13C NMR. At last, these derivatives were evaluated for their anticancer activity using normal human embryonic kidney HEK293 cell and U87-malignant glioma (ATCC-HTB-14) as a cancer cell line. All of the product compounds are active against malignant glioma U87 cell line and show excellent to less antiproliferative activity. Compound 5e (IC50 = 5.75 ± 0.43 μM) among the series shows excellent antiproliferative activity, while 10 compounds (5f, 5b, 5a, 5n, 5r, 5s, 5g, 5q, 5i, and 5j) attributed good activity in the range of IC50 values from 24.17 ± 0.46 to 43.71 ± 0.07 μM. Similarly, all of the compounds obeyed ADMET rules, and some derivatives (5b, 5e, and 5f) are the best toward the inhibition of the CK2 receptor, which give green signal for the treatment of cancer.

Acknowledgments

The authors are thankful to the Higher Education Commission of Pakistan for financial support under NRPU Research Program (No. 14622). The authors are also grateful to the Researchers Supporting Project Number (RSPD2023R1100), King Saud University, Riyadh, Saudi Arabia.

Data Availability Statement

The authors declare no conflict of interest.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c07216.

  • 2D interactions of all of the compounds with casein kinase-2 receptor (Figure S1); drug likeness of the compounds (Table S1); and 1H NMR, 13C NMR, and HR-ESI-MS spectra of the synthetic compounds (Figure S1–S21) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao3c07216_si_001.pdf (5.5MB, pdf)

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Associated Data

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Supplementary Materials

ao3c07216_si_001.pdf (5.5MB, pdf)

Data Availability Statement

The authors declare no conflict of interest.

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